The overall objective of the proposed program is the mechanistic elucidation of the key enzymatic steps in the biosynthesis of carnitine primarily in mammals but secondarily in micro-organisms such as Neurospara and Pseudomonas as well. This objective has significant biochemical and physiological interest because any nutritional or pathological interference with the biosynthesis of carnitine may lead to serious metabolic difficulties specifically with regard to lipids but secondarily with regards to other constituents of the body. In the framework of that objective we shall first concentrate our efforts on determining the mechanism of the last step in carnitine biosynthesis, namely the hydroxylation of gamma-butyrobetaine to camitine by what is known to be an alpha-ketoglutarate-dependent dioxygenase that requires the participation of molecular oxygen, ferrous ions and a reducing agent such as ascorbic acid. We have already developed an assay based on the detritiation of tritiated alpha-butyrobetaine. Using that assay, the purification of the hydroxylase from calf liver will be monitored. Ultimately the purified enzyme will be studied for kinetics and mechanism of action. That hydroxylation occurs stereospecifically with retention of configuration, has already been established, however whether a peroxy or other kind of derivative intervenes will now also be studied. By use of substrate amounts of enzyme we shall attempt to uncouple the decarboxylation of alpha-ketoglutarate from the hydroxylation of gamma-butyrobetaine. We shall try to determine catalase and other proteins enhance the hydroxylation. The hydroxylation by a similar dioxygenase of 6-Trimethyl-L-lysine to 3-hydroxy-6-trimethyl-L-lysine, and the enzymology of its cleavage and subsequent oxidation leading to the formation of gamma-butyro-betaine will be investigated. Finally, we shall determine whether any of these reactions proceed in cultured cells such as fibroblasts and smooth muscle cells or whether these cells depend on the serum in the medium for their carnitine requirement.